Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the (R)_P and (S)_P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. (R)_P-Fonofos and (S)_P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the (S)_P-oxon was a more potent inhibitor than the (R)_P-oxon with k₁ ratios of $\text{(S)}{}_{\mathrm{<mtext>P</mtext>}}\text{(R)}{}_{\mathrm{<mtext>P</mtext>}}$ ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.     

Structure and insecticidal activity of picrotoxinin analogs

Picrotoxinin, a convulsant in mammals, was nontoxic when topically applied on whole house flies. When synergized with piperonyl butoxide, the topical LD₅₀ of picrotoxinin on house flies was 6.50 μg/g or 260 times less toxic on house flies than the synergized insecticide, carbofuran, a carbamate cholinesterase inhibitor.When perfused on the isolated thoracic ganglion of house fly, picrotoxinin was less potent in producing convulsions than carbofuran; however, when assayed on the desheathed thoracic ganglion, picrotoxin was more potent than carbofuran. This suggested a substantial barrier to picrotoxinin preventing diffusion into the house fly central nervous system.Of several picrotoxinin analogs synthesized the 2-iodo,4-isopropyl, 6-methylenebromidecyclohexane-γ-lactone was the most potent convulsant.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

CNS insensitivity to pyrethroids in the resistant kdr strain of house flies

Solutions of tetramethrin, RU 11679, or cismethrin caused uncoupled convulsions in 30–40 min in exposed thoracic ganglia from S_NAIDM house flies at concentrations down to 10^?10M: whereas these same compounds at 10^?6M concentrations failed to produce poisoning symptoms when perfused onto the exposed ganglia of the kdr strain of house fly. The pyrethroid analogs examined had a negative temperature coefficient of action on the exposed thoracic ganglia from S_NAIDM flies. DDT and GH-74 possessed positive temperature coefficients of action on the exposed thoracic ganglion of susceptible house flies. It is concluded that the central nervous system of the kdr strain of house fly is resistant to pyrethroid action; furthermore, the resistance appears to be widespread throughout the house fly nervous system, involving sensory, motor, and central neural elements.     

In vivo measurement of house fly temperature,flight muscle potentials,heartbeat and locomotion during insecticide poisoning

The average heartbeat rate of female adult Musca domestica was near 250 beats/min in vivo at 23°C. However, standard deviation values ranged from ±35 to ±60 depending on the individual house fly. Heartbeat rates in tethered house flies fluctuated between cessation to over 300 beats/min. The heartbeat rate was temperature dependent with a Q₁₀ of 2.3. Either a bite by the Lynx spider, Peucetia vividans (Hentz), or severing the abdomen from the thorax caused the heartbeat to become extremely steady at near 300 beats/min which gradually decreased over several minutes.Application of lethal doses of Monitor or Lindane to the house fly caused thoracic temperature to increase by at most 3°C in conjunction with increased convulsive activity and increased average heartbeat rate. In late stages of poisoning, the heartbeat was relatively uniform indicating a disruption in cardioregulatory nervous activity. Response of the house fly to carbofuran or its N-thiomethyl analog was similar to that of Monitor and Lindane except in late stages of poisoning where the heartbeat continued to exhibit large variations in average rate.     

Neural and behavioral correlates of pyrethroid and DDT-type poisoning in the house fly, Musca domestica L

Intact house flies were observed during poisoning caused by several pyrethroid and DDT-type insecticides. The two insecticide classes could be generally distinguished from each other based on differences in symptoms and several physiological correlates. Both insecticide types caused motor unit repetitive backfiring, but the temporal development and stability of repetitiveness were distinctly different between the two classes. Repetitive backfiring always disappeared at low temperatures, but DDT-type backfiring disappeared at lower temperatures than the pyrethroids. trans-Tetramethrin caused a threshold increase in flight motor nerve endings which did not occur in DDT or trans-Barthrin poisoning. Pyrethroids caused “uncoupling” of the flight motor pattern, while DDT-types did not. trans-Barthrin, a methylenedioxyphenyl pyrethroid, was unique in causing both symptoms and physiological aberrations which more closely resembled those of the DDT-types than the pyrethroids.     

Metabolism of O,S-dimethyl propionyl- and hexanoylphosphoramidothioate in the house fly and white mouse

The metabolism of O,S-dimethyl propionyl- and hexanoylphosphoramidothioate was investigated in the white mouse and house flies. Compared to the hexanoylphosphoramidothioate, the propionyl analog is approximately 35-fold more toxic to house flies and is 10-fold less toxic to mice. On a percentage basis, substantially larger amounts of methamidophos were detected in house flies treated topically with the propionylphosphoramidothioate than in flies treated with the hexanoyl derivative. The reverse was evident in the case of the mouse where much larger amounts of methamidophos were formed after oral treatment with the hexanoylphosphoramidothioate. Minor amounts of other metabolic products also were detected, including an unknown from the hexanoylphosphoramidothioate. Metabolism of the S-methyl moiety to carbon dioxide appeared to be a major pathway for metabolic degradation of both compounds in both the white mouse and house fly. The difference in toxicity of the two acylphosphoramidothioates to the mouse and house fly is attributed to difference in the amounts of methamidophos formed in the animals.     

Toxicokinetics of carbofuran in house fly, Musca domestica L

The kinetics of accumulation and elimination of lethal doses of [¹⁴C]carbofuran in the hemolymph of the house fly suggest a one-compartment open model. Carbofuran in the hemolymph appeared to be in equilibrium with that in the tissues very soon after treatment.Following topical application of carbofuran, the rate of onset of symptoms of poisoning was correlated with the amount of carbofuran in the hemolymph, and the onset of convulsions only occurred after the concentration of carbofuran in the hemolymph reached μM levels. This value correlated well with neurobioassays of known concentrations of carbofuran perfused in saline onto the isolated thoracic ganglion.Following topical doses, carbofuran concentration in the hemolymph reached a peak within an hour and then gradually declined. At an LD₆₀ dose, the initial decline in carbofuran concentration in the hemolymph over time was significantly slower than the decline after an LD₁₀ dose, suggesting saturation kinetics.Hemolymph was collected from house flies for up to 3 hr following topical application of toxic amounts of carbofuran. Thereafter, hemolymph volume decreased and blood samples could not be collected. Curiously, hemolymph samples could be collected for 5 hr from house flies that were injected with toxic doses of carbofuran.     

Penetration and metabolism of 2-isopropoxyphenyl N-methyl-N-(2-methyl-4-tert-butylphenylsulfenyl) carbamate in the house fly and honeybee

The penetration and metabolism of 2 - isopropoxyphenyl N-methyl-N-(2-methyl-4-tert - butylphenylsulfenyl)carbamate or sulfenyl-propoxur was examined in the house fly and honeybee. Reduced penetration was found to be a factor contributing to the lower toxicity of sulfenyl-propoxur to the honeybee. Honeybees and house flies metabolized sulfenyl-propoxur qualitatively in a similar manner. Quantitatively, larger amounts of propoxur were found in the house fly than in the honeybee soon after treatment with sulfenyl-propoxur. The slower rate of conversion of sulfenyl-propoxur to propoxur was considered as another factor responsible for the lower toxicity of the sulfenylated derivative to bees. The high susceptibility of bees to propoxur was related to high internal amounts of unchanged propoxur found soon after treatment.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

Toxicity of spinosad to susceptible and resistant strains of house flies,Musca domestica

The toxicity of spinosad, a new insecticide derived from the bacterium Saccharopolyspora spinosa, was evaluated against susceptible and resistant strains of house fly (Musca domestica L.). Spinosad was highly toxic to house flies based on 72-h LD₅₀ values and the symptoms of poisoning were consistent with a neurotoxic mechanism of action. Spinosad was relatively slow acting, with the maximum toxicity noted at 72 h. Piperonyl butoxide and S,S,S,-tribu-tylphosphorotrithioate synergized the toxicity of spinosad by 3·0- and 1·8-fold, respectively, while diethyl maleate had no significant effect. These results suggest that there is a small degree of monooxygenase-mediated spinosad detoxification in house flies, while hydrolases may be only minimally important and glutathione transferases may have no role. There were no substantial levels of cross-resistance detected, except in the LPR strain where a low 4·3-fold cross-resistance was observed. The cyclodiene-resistant OCR strain was 2·7-fold more sensitive to spinosad than the susceptible strain (CS). These results suggest that cross-resistance may not be a limiting factor for the use of spinosad against house flies. © 1998 Society of Chemical Industry     

Behavioural and electrophysiological investigation of 1-(7-ethoxygeranyl)-2-methylbenzimidazole on the house fly Musca domestica

The insecticidal properties of 1-(7-ethoxygeranyl)-2-methylbenzimidazole (EGMB) were investigated on larval and adult house flies. Unsynergised EGMB gave topical LD₅₀ values of 0.53 μg per female fly on NAIDM strain house flies. When flies were pretreated with 5.2 μg piperonyl butoxide, susceptibility was increased (LD₅₀ 0.12 μg per female fly). House fly larvae were less susceptible to EGMB (LD₅₀ 2.2 μg). Poisoning with EGMB resulted in a rapid reduction in locomotor activity of both larval and adult house flies. This reduction in locomotion was progressive and led to complete paralysis. Various parameters of larval nervous system function were investigated in larvae during these early phases of poisoning. As early as 15 min after dosing larvae with LD₉₅ doses of EGMB, sensory nerves were less responsive. Over a somewhat longer time (2–4 h), neurally evoked contractures were adversely affected by EGMB. In some cases, this effect appeared to be due to reduced postsynaptic potential amplitude; in other instances, it appeared to be due to an effect independent of neuromuscular transmission. The close temporal correlation between behavioural and electrophysiological observations suggests that the nervous and muscular systems are important sites of action of EGMB.     

The effect of phenobarbital induction on glutathione conjugation of diazinon in susceptible and resistant house flies

The relationship between glutathione S-transferase activity toward 3,4-dichloronitrobenzene and O-alkyl or O-aryl conjugation of diazinon was investigated in eight strains of house flies. No significant difference was found in the amount of O-aryl conjugation. In contrast, house flies which had higher glutathione S-transferase activity toward 3,4-dichloronitrobenzene also had higher O-alkyl conjugating activity toward diazinon. The glutathione S-transferase(s) in phenobarbital-pretreated flies degraded diazinon faster than those in the nontreated ones. The present results showed that the formation of the O-alkyl conjugate was enhanced by phenobarbital pretreatment, while the formation of the O-aryl conjugate was not affected by induction. Based on these findings, it would appear that one of the multiple forms of glutathione S-transferase is specifically induced and responsible for the increase in O-alkyl conjugation.     

Metabolism of O,O-dimethyl S-[α-carboethoxy)benzyl]phosphorodithioate (phenthoate) in the white mouse and house flies

The metabolism of O,O-dimethyl S-[α-(carboethoxy)benzyl]phosphorodithioate (phenthoate), an organophosphorus insecticide of low mammalian toxicity, was investigated in white mice and in susceptible and resistant strains of house flies. Phenthoate was metabolized rapidly in the mouse to a wide variety of detoxication products and only an insignificant amount of phenthoate oxon was detected. The same detoxication products were produced in house flies but, compared to the mouse, substantial amounts of phenthoate oxon also were found. The selective toxicity of phenthoate between insect and mammal is attributable to the difference in the accumulation of the oxon.     

Comparative ester hydrolysis of carbaryl and ethiofencarb in four mammalian species

The comparative ester hydrolysis and selective toxicity of carbamate insecticides were studied in four mammalian species. Hydrolysis rates of carbaryl and ethiofencarb (Croneton) were examined in the rat, mouse, guinea pig, and gerbil. Respiratory ¹⁴CO₂ resulting from the hydrolysis of orally administered [carbonyl-¹⁴C]carbamates (0.2 mg/kg) was taken as measure of in vivo hydrolytic capabilities. Ester hydrolysis was found to be greater for ethiofencarb than for carbaryl in all species tested, although the relative order of hydrolysis among species was the same with both compounds. After 24 hr, gerbils had hydrolyzed 91% of the ethiofencarb and 65% of the carbaryl. Guinea pigs hydrolyzed somewhat less of the compounds, 65 and 58%, but considerably more than rats and mice, about 40 and 25%. Comparing hydrolysis capabilities to acute toxicity data revealed that those species exhibiting the greatest hydrolysis were equally or more susceptible to carbamate poisoning than those having lesser hydrolytic capabilities. While ester hydrolysis destroys the anticholinesterase activity of carbamates, it is clear from these findings that factors other than hydrolysis are largely responsible for the variation in toxicity of the carbamates to different mammalian species.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

Acetylcholinesterase isozymes of the house fly thorax: In vivo inhibition by organophosphorous insecticides

Polyacrylamide gel electrophoresis of the supernatant fraction of house fly thoracic homogenates demonstrated five electrophoretic bands having cholinesterase activity. The five esteratic bands were considered to be isozymes of acetylcholinesterase based on their staining properties with acetylthiocholine. There appeared to be no visual or densitometric difference in the intensity of staining of the isozymes when acetylthiocholine was compared with butyrylthiocholine as substrate. Selective inhibition of these isozymes in vivo by organophosphate insecticides may contribute only in small part to the mode of action of organophosphate insecticides.     

The A302S mutation in Rdl that confers resistance to cyclodienes and limited cross-resistance to fipronil is undetectable in field populations of house flies from the USA

Fipronil is a relatively new insecticide with great potential for insect control, however widespread use of cyclodiene insecticides has selected for an A302S mutation in the Rdl (GABA gated chloride channel) allele. This mutation gives resistance to cyclodienes and limited cross-resistance to fipronil. Given the concern over the possible reduction in efficacy and/or lifetime that fipronil might be used for pest control (given the extensive use of cyclodienes in the past), it is important to know the frequency of the A302S Rdl mutation in field populations. To ascertain the relative frequency of the A302S Rdl mutation in house fly populations we used three experimental approaches. First, we attempted to select for fipronil resistance by initially treating 33,100 field collected flies and selecting 14 additional generations. We were unable to produce a highly resistant strain. Second, we directly sequenced field collected flies. Third, we tested field collected house flies with a diagnostic dose of dieldrin and then genotyped the survivors. Out of the 4750 flies tested, there were no Rdl resistance alleles detected. We conclude that the resistant Rdl allele is rare in house flies in the US due to decades without cyclodiene use and a fitness disadvantage (in the absence of cyclodienes) of the 302S Rdl allele. The limited cross-resistance provided by the cyclodiene resistant Rdl allele, combined with the very low frequency of this allele in field populations, suggests that fipronil could be a promising insecticide for house fly control.     

Metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-dimethoxyphosphinothioyl-N-methylcarbamate in the house fly,rat, and mouse

The metabolism of a selectively toxic derivative of carbofuran, 2,2-dimethyl-2,3-dihydrobenzofuranyl-7 N-dimethoxyphosphinothioyl N-methylcarbamate (PSC), was examined in the house fly, rat, and mouse. In house flies, PSC is metabolized mainly to carbofuran and related oxidation products containing the intact N-methylcarbamyl ester moiety. Degradation to phenolic products was the principal route of metabolism in rodents. The results indicate that the selective toxicity of PSC between insects and mammals is attributable to differing pathways of metabolism.     

Insecticidal properties,antiesterase activities,and metabolism of methamidophos

Methamidophos is highly toxic to insects but at best is a moderate cholinesterase inhibitor. Evaluation of the kinetics of its housefly cholinesterase inhibition showed that its affinity for the enzyme and its phosphorylation and bimolecular inhibition rates are all relatively low. In vivo metabolism studies in houseflies provided evidence that it is not activated to a more effective cholinesterase inhibitor and indirect evidence also was obtained for its slow degradation. In vitro metabolism studies in housefly and mouse tissues provided additional evidence for its lack of activation and slow metabolic degradation. Compared to other effective organophosphorus insecticides, methamidophos was slow in producing acute symptoms of poisoning and cholinesterase inhibition and required the accumulation of comparatively high internal levels for toxic effects. However, in vivo cholinesterase inhibition studies provided evidence for the interrelationships of cholinesterase inhibition and toxic effects. Thus, its relative stability and low in vivo degradation appeared to be of critical importance in accumulating and maintaining a sufficient internal concentration for a sufficiently long period of time to permit the development of its slowly expressed toxicity.